The present invention relates to an oral solid formulation of compound anti-tubercular drug and preparation method thereof, in particular, to an oral solid formulation of a four-ingredient compound anti-tubercular drug comprising rifampicin, isoniazid, pyrazinamide and ethambutol hydrochloride as active ingredients.
Tuberculosis is a type of chronic infectious diseases caused by tubercle bacillus, which may involve with various tissues and organs in the body, e.g. lung, kidney, intestine, bone etc. Among others, lung tuberculosis is the most frequently found type. Tuberculosis is a major problem influencing the health of people in developing countries. Recently emergence of mycobacterium infections in HIV infected individuals is also on the rise, and becomes a stubborn problem in developed countries. According to the official statistical figures published by World Health Organization (WHO), the number of diagnosis confirmed patients suffering from active tuberculosis in the world is over 20 million, while the number of newly founded patients is up to 8.7 million per year, in which the incidence of lung tuberculosis in Asia occupies 70% of total in the world. India and China occupy the first and second highest incidence of lung tuberculosis in Asia. Antibiotics are commonly used to treat tuberculosis in clinical practice. However, in contrast to treat typical bacterial infections, it requires longer time for the tuberculosis patients to be completely cured, and generally, the treatment will last for about 6-12 months. In view of the clinical treatment status, due to the long period for taking in pills, drug resistance easily generates. To some extent, the phenomenon of drug resistance is increasingly aggravating in the world mainly due to the improper treatment measures carried out, especially due to the single use of one specific kind of antibiotics, or the patient does not normally take in pills according to the prescription, such as missing doses and insufficient dosage. According to “Multidrug and extensively drug-resistant tuberculosis: 2010 global report on surveillance and response. Geneva, Switzerland: World Health Organization, 2010” which is recently finished, it is estimated that 440 thousand people in the world suffer from multidrug-resistant tuberculosis, in which one of the third have died. In view of the drug-resistant phenomenon constantly occurs, WHO and International Union against Tuberculosis and Lung Disease have both announced that it has been in the state of emergency regarding to the treatment of lung tuberculosis. Since the continuing emergence of the drug-resistant phenomenon, the fixed dose combination (FDC method) has been widely used for treating lung tuberculosis. One of the commonly used compound drugs in FDC method is a four-ingredient compound drug, i.e. in addition to the rifampicin(R) and isoniazid(H) which are most efficient for the tuberculosis treatment, it further contains pyrazinamide(Z) and ethambutol hydrochloride(E). Such a four-ingredient compound drug may be a compound drug tablet and capsule, etc, and has been formally approved and accepted by WHO for the tuberculosis treatment. Such a four-compound drug consists of a compound formulation, which increases the compliance of the patients and can reduce the occurrence of drug-resistance to some extent.
Rifampicin(R) is also known as rifampin, which is a semi-synthesized rifamycins derivative. Rifampicin is a brick red color crystal, and has a melting point of 183° C. Rifampicin is poorly soluble in water (solubility: 1.3 mg/ml in water, pH 4.3; 2.5 mg/ml in water, pH 7.3; 100 mg/ml in water, DMSO), and is unstable in acid. Rifampicin can easily react with isoniazid, and also may be oxidized by factors of air, light exposure, etc. The molecule structure of rifampicin is as below:

Rifampicin is an efficient broad-spectrum antibacterial drug, and has a strong effect on inhibiting or eliminating tubercle bacillus. The anti-tubercular effect is only slightly weaker than isoniazid, but is stronger than streptomycin. The minimum inhibitory concentration is about 0.02-0.05 μg/ml. Rifampicin has effect on the bacteria not only in the multiplication stage, but also in the rest stage. Rifampicin is also effective to the strains showing drug-resistance to other anti-tubercular drugs. Rifampicin can eliminate tubercle bacillus in macrophage, fibrotic cavity, caseous focus, and also have strong inhibiting effect on gram positive coccus, such as staphylococcus aureus, streptococcus, and pneumococcus. For gram negative coccus, such as meningococcus, gonococcus, as well as leprosy bacillus, rifampicin also possesses strong inhibiting effect. High concentration of rifampicin inhibits varida virus and chlamydia trachomatis. The single use of rifampicin tends to generate drug-resistance, and thus is usually combined with other first-line pharmaceuticals for the initial treatment and retreatment of patients suffering serious diseases, so as to enhance the efficacy and retard the generation of drug-resistance. There is no cross drug-resistance between rifampicin and other anti-tubercular drugs. Rifampicin may selectively inhibit the RNA polymerase of the bacterial-dependent DNA and block the synthesis of mRNA, but has no effect on the RNA polymerase of animal molecules.
Isoniazid (H) is also known as rimifon, and the chemical name is 4-pyridylcarbonylhydrazine. Isoniazid is easily soluble in water, slightly soluble in ethanol, and very slightly soluble in ethyl ether. The structure of carbonylhydrazine is not stable, and in acidic or basic condition, it can be hydrolyzed to form isonicotinic acid and hydrazine. The free hydrazine increases the toxicity, and light, heavy metals, temperature, pH, etc can accelerate the hydrolysis thereof.
The molecule structure of isoniazid is as below:

Isoniazid has a good bacterial inhibiting effect on tubercle bacillus, and has a good efficacy. The dose of isoniazid is used in a small range, and the toxicity is relatively low, which is easily to be accepted by the patients. The oral absorption rate is 90%, and the serum drug concentration reaches the peak in 1-2 hours after administration. The Vd is 0.61±0.11 L/kg, and the protein binding rate is very low. Isoniazid is mainly used in the progressive stage, dissolving and spreading stage, absorption stage of lung tuberculosis, and also can be used in tubercular meningitis and other extrapulmonary tuberculosis, etc. Isoniazid is usually used in combination with other anti-tubercular drugs in order to enhance the efficacy and overcome the drug-resistant bacteria.
Pyrazinamide (Z) is slightly soluble in water and ethanol, and very slightly soluble in ethyl ether. Pyrazinamide has an effect on accelerating the reaction of rifampicin with isoniazid. The molecule structure of pyrazinamide is as below:

Pyrazinamide has a good bacterial inhibiting effect on human type tubercle bacillus, and shows the largest bacterial eliminating effect at pH 5-5.5. In particular, it is the current best bacterial eliminating drug for tubercle bacillus slowly growing in phagocyte under acidic condition. The minimum inhibitory concentration of pyrazinamide is 12.5 μg/ml. When it reaches a concentration of 50 μg/ml, pyrazinamide can eliminate tubercle bacillus. The intra-cellular concentration of the drug for inhibiting tubercle bacillus is 10 folds lower than the extracellular concentration. It almost has no inhibiting effect under neutral and basic conditions. The mechanism may be involved with pyrazinoic acid. When pyrazinamide penetrates into the phagocyte and into the body of tubercle bacillus, the amidase in the bacterial body deamidates the amide group of pyrazinamide, converts it to pyrazinoic acid, and exerts the bacterial inhibiting effect. In addition, since pyrazinamide is similar in chemical structure with nicotinamide, pyrazinamide interrupts the dehydrogease by substituting nicotinamide, prevents the dehydrogenation effect, and avoids the utilization of oxygen by tubercle bacillus, and thus taking influence on the regular metabolism of the bacteria and causing them to be eliminated.
Ethambutol hydrochloride (E) is easily soluble in water, is easy to absorb moisture, and provides atmosphere for the reaction between rifampicin and isoniazid. The molecule structure of ethambutol hydrochloride is as below:

Ethambutol hydrochloride is suitable for treating tuberculosis in combination with other anti-tubercular drugs. The single use of ethambutol hydrochloride tends to generate drug-resistance. Ethambutol hydrochloride has strong activity for the bacteria in the growth and reproduction stage, but has almost no effect on the bacteria in the resting stage.
RHZE combination has a certain importance in clinical practice, but problem also occurs. First, if rifampicin is in direct contacts with isoniazid, they will tend to react with each other, especially in the stomach acidic condition. This makes the bioavailability of rifampicin in a compound drug lower than that of singly used rifampicin, and thus the treatment effect would be compromised or the lower level of rifampicin can generate drug-resistance for patients. It is reported in references (e.g. see Sosa et al, 2005, Ars Pharm, 46:353-364) that under the condition of gastric acid (pH 1-3), the direct contact between rifampicin and isoniazid is easily to react and generate isoniazone. The experiments conducted by Singh et al (see Singh et al, 2000, Pharm. Pharmacol. Commun. 6: 405-410) also demonstrate the fact. It indicates in the research that under the stomach acidic condition, rifampicin decompose to 3-formyl-rifamycin in the absence of isoniazid. In the presence of isoniazid, the generated 3-formyl-rifamycin rapidly reacts with isoniazid, and forms isoniazone via a second order reaction. Since isoniazone is not stable in the acidic condition, it regenerates 3-formyl-rifamycin and isoniazid via a slow first order reaction in a reversible way. In such a complex reaction, rifampicin is further degraded, while isoniazid is recovered. Although isoniazone has some anti-bacterial activity, the anti-bacterial activity is lower than that of rifampicin. Meanwhile, in RHZE combination, pyrazinamide has catalytic effect on the reaction between rifampicin and isoniazid, and ethambutol hydrochloride, which is easy to absorb moisture, provides conditions for the reaction between rifampicin and isoniazid. These are some of the major factors which cause the formulation unstable, and the bioavailability in a compound drug is lower than that in singly used rifampicin. Second, the solubility of rifampicin is also a problem. Rifampicin is a drug having a low solubility, high hyperosmoticity and is easier to dissociate. Its solubility is pH dependent, and shows a large solubility difference in different pH conditions in gastrointestinal tract. Some experiments indicate that at pH 1.4, the solubility reaches about 125 mg/ml, and 80-90% dissolves in 10 minutes. But at pH>3, the solubility is less than 6 mg/ml. If a simple enteric coating method is applied to render rifampicin not to release in stomach, but release in intestinal tract instead, the insolubility of rifampicin in intestinal tract also will directly lead to a decrease of the bioavailability of rifampicin.
Therefore, the problem to be solved by many drug research and development institutions and manufacturers is how to improve the bioavailability of rifampicin in four-ingredient (RHZE) compound formulations. In this regard, some related researches have been reported. For example, WO02/11728 discloses that rifampicin and a pH-dependent carrier are dissolved in media to prepare solid dispersions, so as to increase the solubility of rifampicin. However, the present inventors found in our experiments that since the amount of pH-dependent carrier used is relative low, the large amount release of rifampicin in acidic condition is difficult to control. The patent cannot achieve the same effect of the solid formulation as in the present invention with respect to the bioavailability of rifampicin in RHZE compound drug. The rifampicin solid dispersion in the inventive solid formulation not only increases the solubility and dissolution rate of rifampicin, but also ensures less release or no release of rifampicin in the acidic stomach condition and thus the reduction of the reaction between rifampicin and isoniazid in the body. Meanwhile, the fact that the excipients reduce contact of rifampicin with isoniazid in the formulation ensures the stability of the formulation during storage. The in-vivo and in-vitro stability of rifampicin in the RHZE compound drug facilitates to improve the bioavailability of rifampicin in the compound drug, ensures the therapeutic efficacy and reduces the generation of drug-resistance.